THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Ligand‐gated ion channels

The Concise Guide to PHARMACOLOGY 2017/18 provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13879/full. Ligand‐gated ion channels are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, voltage‐gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

loop receptors due to the presence of a defining loop of residues formed by a disulphide bond in the extracellular domain of their constituent subunits [259,353]. However, the prokaryotic ancestors of these receptors contain no such loop and the term pentameric ligand-gated ion channel (pLGIC) is gaining acceptance in the literature [145]. The ionotropic glutamate and P2X receptors are tetrameric and trimeric structures, respectively. Multiple genes encode the subunits of LGICs and the majority of these receptors are heteromultimers. Such combinational diversity results, within each class of LGIC, in a wide range of receptors with differing pharmacological and biophysical properties and varying patterns of expression within the nervous system and other tissues. The LGICs thus present attractive targets for new therapeutic agents with improved discrimination between receptor isoforms and a reduced propensity for off-target effects. The development of novel, faster screening techniques for compounds acting on LGICs [100] will greatly aid in the development of such agents.

Comments
ASIC1a and ASIC1b are also blocked by diarylamidines (IC 50˜3 μM for ASIC1a) ASIC2 is also blocked by diarylamidines Comments ASIC3 is also blocked by diarylamidines Comments: psalmotoxin 1 (PcTx1) inhibits ASIC1a by increasing the affinity to H+ and promoting channel desensitization [64,107]. PcTx1 has little effect on ASIC2a, ASIC3 or ASIC1a expressed as a heteromultimer with either ASIC2a, or ASIC3 but does inhibit ASIC1a expressed as a heteromultimer with ASIC2b [330]. ASIC1-containing homo-and heteromers are inhibited by Mambalgins, toxins contained in the black mamba venom, which induce in ASIC1a an acidic shift of the pH dependence of activation [89]. APETx2 most potently blocks homomeric ASIC3 channels, but also ASIC2b+ASIC3, ASIC1b+ASIC3, and ASIC1a+ASIC3 heteromeric channels with IC 50 values of 117 nM, 900 nM and 2 μM, respectively. APETx2 has no effect on ASIC1a, ASIC1b, ASIC2a, or ASIC2a+ASIC3 [88,90]. APETx2 inhibits however also voltage-gated Na+ channels [34,297]. IC 50 values for A-317567 are inferred from blockade of ASIC channels native to dorsal root ganglion neurones [99]. The pEC 50 values for proton activation of ASIC channels are influenced by numerous factors including extracellular di-and poly-valent ions, Zn 2+ , protein kinase C and serine proteases (reviewed in [193,382]). Rapid acidification is required for activation of ASIC1 and ASIC3 due to fast inactivation/desensitization. pEC 50 values for H + -activation of either transient, or sustained, currents mediated by ASIC3 vary in the literature and may reflect species and/or methodological differences [16,79,375]. The transient ASIC cur-rent component is Na + -selective (PNa/PK of about 10) [375,392] whereas the sustained current component that is observed with ASIC3 and some ASIC heteromers is non-selective between Na + and K + [79]. The reducing agents dithiothreitol (DTT) and glutathione (GSH) increase ASIC1a currents expressed in CHO cells and ASIC-like currents in sensory ganglia and central neurons [8,68] whereas oxidation, through the formation of intersubunit disulphide bonds, reduces currents mediated by ASIC1a [405]. ASIC1a is also irreversibly modulated by extracellular serine proteases, such as trypsin, through proteolytic cleavage [373]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are direct inhibitors of ASIC currents (reviewed in [22]). Extracellular Zn 2+ potentiates Searchable database: http://www.guidetopharmacology.org/index.jsp Acid-sensing (proton-gated) ion channels (ASICs) S134 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13879/full proton activation of homomeric and heteromeric channels incorporating ASIC2a, but not homomeric ASIC1a or ASIC3 channels [23]. However, removal of contaminating Zn 2+ by chelation reveals a high affinity block of homomeric ASIC1a and heteromeric ASIC1a+ASIC2 channels by Zn 2+ indicating complex biphasic actions of the divalent [69]. Nitric oxide potentiates submaximal currents activated by H + mediated by ASIC1a, ASIC1b, ASIC2a and ASIC3 [47]. Ammonium ions activate ASIC channels (most likely ASIC1a) in midbrain dopaminergic neurones: that may be relevant to neuronal disorders associated with hyperammonemia [302]. The positive modulation of homomeric, heteromeric and native ASIC channels by the peptide FMRFamide and related substances, such as neuropeptides FF and SF, is reviewed in detail in [369]. Inflammatory conditions and particular proinflammatory mediators such as arachidonic acid induce overexpression of ASIC-encoding genes and enhance ASIC currents [85,241,337]. The sustained current component mediated by ASIC3 is potentiated by hypertonic solutions in a manner that is synergistic with the effect of arachidonic acid [85]. ASIC3 is partially activated by the lipids lysophosphatidylcholine (LPC) and arachidonic acid [244]. Mit-Toxin, which is contained in the venom of the Texas coral snake, activates several ASIC subtypes [37]. Selective activation of ASIC3 by GMQ at a site separate from the proton binding site is potentiated by mild acidosis and reduced extracellular Ca 2+ [402]. Epithelial sodium channels (ENaC) Ligand-gated ion channels → Epithelial sodium channels (ENaC) Overview: The epithelial sodium channels (ENaC) mediate sodium reabsorption in the aldosterone-sensitive distal part of the nephron and the collecting duct of the kidney. ENaC is assembled as a heterotrimer composed of three subunits α, β, and γ or δ, β, and γ [137]. These subunits consitute a family within the ENaC/Degenerin super-family [137]. Genes encoding ENaC subunits are found in all vertebrates with the exception of ray-finned fishes [137]. ENaC composed of α, β, and γ subunits is located mostly in tight or high-resistance epithelial tissues such as the airways, distal colon and exocrine glands [104]. ENaC activity is tightly regulated in the kidney by aldosterone, angiotensin II (AGT, P01019), vasopressin (AVP, P01185), insulin (INS, P01308) and glucocorticoids; this fine regulation of ENaC is essential to maintain sodium balance between daily intake and urinary excretion of sodium, circulating volume and blood pressure. ENaC expression is also vital for clearance of foetal lung fluid, and to maintain air-surface-liquid [160,227]. Sodium reabsorption is suppressed by the 'potassium-sparing' diuretics amiloride and triamterene. ENaC is a heteromultimeric channel made of homologous α β and γ subunits. The primary structure of the α ENaC subunit was identified by expression cloning [48,137]; β and γ ENaC subunits were identified by functional complementation of the α subunit [49,137]. Each ENaC subunit contains 2 TM α helices connected by a large extracellular loop and short cytoplasmic amino-and carboxy-termini. The stoichiometry of the epithelial sodium channel in the kidney and related epithelia is, by homology with the structurally related channel ASIC1a, thought to be a heterotrimer of 1α:1β:1γ subunits [125]. Channel blockers P552-02 (pIC 50 8.1), benzamil (pIC 50 ∼8), amiloride (pIC 50 6.7-7), triamterene (pIC 50 ∼5.3) [49,192] Functional Characteristics γ ≈ 4-5 pS, P Na /P K > 20; tonically open at rest; expression and ion flux regulated by circulating aldosterone-mediated changes in gene transcription. The action of aldosterone, which occurs in 'early' (1.5-3 h) and 'late' (6-24 hr) phases is competitively antagonised by spironolactone, its active metabolites and eplerenone. Glucocorticoids are important functional regulators in lung/airways and this control is potentiated by thyroid hormone; but the mechanism underlying such potentiation is unclear [19,311,322]. The density of channels in the apical membrane, and hence G Na , can be controlledvia both serum and glucocorticoid-regulated kinases (SGK1, 2 and 3) [80,114] and via cAMP/PKA [267]; and these protein kinases appear to act by inactivating Nedd-4/2, a ubiquitin ligase that normally targets the ENaC channel complex for internalization and degradation [35,80]. ENaC is constitutively activated by soluble and membrane-bound serine proteases, such as furin, prostasin (CAP1), plasmin and elastase [202,203,305,313,314]. The activation of ENaC by proteases is blocked by a protein, SPLUNC1, secreted by the airways and which binds specifically to ENaC to prevent its cleavage [120]. Pharmacological inhibitors of proteases (e.g. camostat acting upon prostasin) reduce the activity of ENaC [237]. Phosphatidylinositides such as PtIns(4,5)P 2 and PtIns(3,4,5)P 3 ) stabilise channel gating probably by binding to the β and γ ENaC subunits, respectively [235,307], whilst C terminal phosphorylation of β and γ-ENaC by ERK1/2 has been reported to inhibit the withdrawal of the channel complex from the apical membrane [393]. This effect may contribute to the cAMP-mediated increase in sodium conductance.  [137]. Liddle's syndrome (including features of salt-sensitive hypertension and hypokalemia), is associated with gain of function mutations in the β and γ subunits leading to defective ENaC ubiquitylation and increased stability of active ENaC at the cell surface [137,314,324,343]. Enzymes that deubiquitylate ENaC increase its function in vivo. Pseudohypoaldosteronism type 1 (PHA-1) can occur through either mutations in the gene encoding the mineralocorticoid receptor, or loss of function mutations in genes encoding ENaC subunits [39,137]. Regulation of ENaC by phosphoinositides may underlie insulin ( INS, P01308)-evoked renal Na + retention that can complicate the clinical management of type 2 diabetes using insulin-sensitizing thiazolidinedione drugs [132].

Further reading on Epithelial sodium channels (ENaC)
Boscardin E et al.

GABA A receptors
Ligand-gated ion channels → GABA A receptors Overview: The GABA A receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT 3 and strychnine-sensitive glycine receptors. GABA A receptormediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABA A , slow' [51]. GABA A receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one , one π and one θ GABA A receptor subunits have been reported in mammals [286,287,331,333]. The π -subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4and α6-(both not functional) α5-, β2-, β3and γ2), along with RNA editing of the α3 subunit [75]. The three ρ-subunits, (ρ1-3) function as either homo-or hetero-oligomeric assemblies [60,406]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABA C receptors [406], but they are classified as GABA A receptors by NC-IUPHAR on the basis of structural and functional criteria [20, 286, 287]. Many GABA A receptor subtypes contain α-, βand γ-subunits with the likely stoichiometry 2α.2β.1γ [206,287]. It is thought that the majority of GABA A receptors harbour a single type of αand β -subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABA A receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4-α5-or α6-subunit, or the β1-, γ1-, γ3-, δ-, -and θ -subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6and δ-subunits in cerebellar granule cells, or an α4and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [27,109,265,327,338]. GABA binding occurs at the β+/αsubunit interface and the homologous γ+/αsubunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/βinterface ( [310]; reviewed by [332]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4or α6-subunits are not recognised by 'classical' benzodiazepines, such as flunitrazepam (but see [400]). The trafficking, cell surface expression, internalisation and function of GABA A receptors and their subunits are discussed in detail in several recent reviews [66,166,232,371] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the d subunit appear to be exclusively extrasynaptic. NC-IUPHAR [20, 287] class the GABA A receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABA A receptors are classed as conclusively identified (i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [286,287]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABA A receptor isoforms in detail; such information can be gleaned in the reviews [20,117,181,206,209,270,286,287, 331] and [11,12]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [60,182,274,406]. Comments Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208] Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208] Nomenclature Comments Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208] diazepam and flunitrazepam are not active at this subunit. Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208]. Comments Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208] diazepam and flunitrazepam are not active at this subunit. Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208]. Comments Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208] Nomenclature

Nomenclature
Channel blockers TBPS, picrotoxin TBPS, picrotoxin TBPS, picrotoxin Comments Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [208]  Comments bicuculline is not active at this subunit bicuculline is not active at this subunit bicuculline is not active at this subunit

Comments:
The potency and efficacy of many GABA agonists vary between GABA A receptor isoforms [117,188,209]. For example, gaboxadol is a partial agonist at receptors with the subunit composition α4β3γ2, but elicits currents in excess of those evoked by GABA at the α4β3δ receptor where GABA itself is a low efficacy agonist [32,43]. The antagonists bicuculline and gabazine differ in their ability to suppress spontaneous openings of the GABA A receptor, the former being more effective [359]. The presence of the γ subunit within the heterotrimeric complex reduces the potency and efficacy of agonists [347]. The GABA A receptor contains dis-tinct allosteric sites that bind barbiturates and endogenous (e.g., 5α-pregnan-3α-ol-20-one) and synthetic (e.g., alphaxalone) neuroactive steroids in a diastereo-or enantio-selective manner [28,143,154,368]. Picrotoxinin and TBPS act at an allosteric site within the chloride channel pore to negatively regulate channel activity; negative allosteric regulation by γ-butyrolactone derivatives also involves the picrotoxinin site, whereas positive allosteric regulation by such compounds is proposed to occur at a distinct locus. Many intravenous (e.g., etomidate, propofol) and inhalational (e.g., halothane, isoflurane) anaesthetics and alcohols also exert a regulatory influence upon GABA A receptor activity [38,285]. Specific amino acid residues within GABA A receptor αand β-subunits that influence allosteric regulation by anaesthetic and non-anaesthetic compounds have been identified [141,154]. Photoaffinity labelling of distinct amino acid residues within purified GABA A receptors by the etomidate derivative, [ 3 H]azietomidate, has also been demonstrated [221] and this binding subject to positive allosteric regulation by anaesthetic steroids [220]. An array of natural products including flavonoid and terpenoid compounds exert varied actions at GABA A receptors (reviewed in detail in [181]). In addition to the agents listed in the table, modulators of GABA A receptor activity that exhibit subunit dependent activity include: salicylidene salicylhydrazide [negative allosteric modulator selective for β1versus β2-, or β3-subunit-containing receptors [360]]; fragrent dioxane derivatives [positive allosteric modulators selective for β1versus β2-, or β3-subunit-containing receptors [328]]; loreclezole, etomidate, tracazolate, mefenamic acid, etifoxine, stiripentol, valerenic acid amide [positive allosteric modulators with selectivity for β2/β3over β1-subunit-containing receptors [112,198,206]]; tracazolate [intrinsic efficacy, i.e., potentiation, or inhibition, is dependent upon the identity of the γ1-3-, δ-, or -subunit co-assembed with α1and β1-subunits [358]]; amiloride [selective blockade of receptors containing an α6-subunit [115]]; furosemide [selective blockade of receptors containing an α6subunit co-assembled with β2/β3-, but not β1-subunit [206]]; La 3+ [potentiates responses mediated by α1β3γ2L receptors, weakly inhibits α6β3γ2L receptors, and strongly blocks α6β3δ and α4β3δ receptors [43,321]]; ethanol [selectively potentiates responses mediated by α4β3δ and α6β3δ receptors versus receptors in which β2 replaces β3, or γ replaces δ [378], but see also [205]]; DS1 and DS2 [selectively potentiate responses mediated by δ-subunitcontaining receptors [374]]. It should be noted that the apparent selectivity of some positive allosteric modulators (e.g., neurosteroids such as 5α-pregnan-3α-ol-20-one for δ-subunit-containing receptors (e.g., α1β3δ) may be a consequence of the unusually low efficacy of GABA at this receptor isoform [27,32].

Glycine receptors
Ligand-gated ion channels → Glycine receptors Overview: The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmittergated ion channels that includes the zinc activated channels, GABA A , nicotinic acetylcholine and 5-HT 3 receptors [233]. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [30,129], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1 INS and α1 del ), α2 (α2A and α2B), α3 (α3S and α3L) and β (β 7) subunits and by mRNA editing of the α2 and α3 subunit [103,249,284]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the mature form of the receptor contains α1 (or α3) and β subunits while the immature form is mostly composed of only α2 subunits. RNA transcripts encoding the α4-subunit have not been detected in adult humans. The N-terminal domain of the αsubunit contains both the agonist and strychnine binding sites that consist of several discontinuous regions of amino acids. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. The latter is a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors heterooligomeric receptors to the synapse [201,204,268 Selective agonists (potency order)   [394]. In addition, potentiation of glycine receptor activity by cannabinoids has been claimed to contribute to cannabis-induced analgesia relying on Ser296/307 (α1/α3) in M3 [389]. Several analogues of muscimol and piperidine act as agonists and antagonists of both glycine and GABA A receptors. Picrotoxin acts as an allosteric inhibitor that appears to bind within the pore, and shows strong selectivity towards homomeric receptors. While its components, picrotoxinin and picrotin, have equal potencies at α1 receptors, their potencies at α2 and α3 receptors differ modestly and may allow some distinction between different receptor types [395]. Binding of picrotoxin within the pore has been demonstrated in the crystal structure of the related C. elegans GluCl Cys-loop receptor [144]. In addition to the compounds listed in the table, numerous agents act as allosteric regulators of glycine receptors (comprehensively reviewed in [215,234,381,399]). Zn 2+ acts through distinct binding sites of high-and lowaffinity to allosterically enhance channel function at low (<10 μM) concentrations and inhibits responses at higher concentrations in a subunit selective manner [258]. The effect of Zn 2+ is somewhat mimicked by Ni 2+ . Endogenous Zn 2+ is essential for normal glycinergic neurotransmission mediated by α1 subunitcontaining receptors [147]. Elevation of intracellular Ca 2+ produces fast potentiation of glycine receptor-mediated responses.
Dideoxyforskolin (4 μM) and tamoxifen (0.2-5 μM) both potentiate responses to low glycine concentrations (15 μM), but act as inhibitors at higher glycine concentrations (100 μM). Additional modulatory agents that enhance glycine receptor function include inhalational, and several intravenous general anaesthetics (e.g. minaxolone, propofol and pentobarbitone) and certain neurosteroids. Ethanol and higher order n-alcohols also enhance glycine receptor function although whether this occurs by a direct allosteric action at the receptor [245], or through βγ subunits [396] is debated. Recent crystal structures of the bacterial homologue, GLIC, have identified transmembrane binding pockets for both anaesthetics [282] and alcohols [156]. Solvents inhaled as drugs of abuse (e.g. toluene, 1-1-1-trichloroethane) may act at sites that overlap with those recognising alcohols and volatile anaesthetics to produce potentiation of glycine receptor function. The function of glycine receptors formed as homomeric complexes of α1 or α2 subunits, or hetero-oligomers of α1/β or α2/β subunits, is differentially affected by the 5-HT 3 receptor antagonist tropisetron (ICS 205-930) which may evoke potentiation (which may occur within the femtomolar range at the homomeric glycine α1 receptor), or inhibition, depending upon the subunit composition of the receptor and the concentrations of the modulator and glycine employed. Potentiation and inhibition by tropeines involves different binding modes [238]. Additional tropeines, including atropine, modulate glycine receptor activity.

Ionotropic glutamate receptors
Ligand-gated ion channels → Ionotropic glutamate receptors Overview: The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [86,226,365]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), three transmembrane domains composed of three membrane spans (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy-terminal domain (CTD) [184,211,246,271,365]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2 -see below) has recently been solved at 3.6Å resolution [340] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA re-ceptors [187]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [62,74,86,106,171,177,195,288,289,290,365,388]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.
The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [71]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.

AMPA and Kainate receptors
AMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 and GluD1, GluD2, GluK1, GluK2, GluK3, GluK4 and GluK5 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [108,165,260,362]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [218,300,303]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [171,300,303]). Kainate receptors may also exhibit 'metabotropic' functions [218,312]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [219,300]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl-for their activation [40,306]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca 2+ ; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and Joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [163,326]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [218,300]. Native AMPA and kainate receptors displaying differential channel conductances, Ca 2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [73, 163,224]. GluA1-4 can exist as two variants generated by alternative splicing (termed 'flip' and 'flop') that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [260]. Splice variants of GluK1-3 also exist which affects their trafficking [218,300].  --

NMDA receptors
NMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2, GluN3 and GluN4 subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [63,105]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [119,187,246]. However, more complex tri-heteromeric assem-blies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generatedin vitro and occurin vivo. The NMDA receptor channel commonly has a high relative permeability to Ca 2+ and is blocked, in a voltage-dependent manner, by Mg 2+ such that at resting potentials the response is substantially inhibited.   [96], Mg 2+ (GluN2A = GluN2B > GluN2C = GluN2D),  [62,96,106,212,290,365]. In addition to the glutamate and glycine binding sites documented in the table, physiologically important inhibitory modulatory sites exist for Mg 2+ , Zn 2+ , and protons [74, 86,365]. Voltage-independent inhibition by Zn 2+ binding with high affinity within the ATD is highly subunit selective (GluN2A GluN2B > GluN2C ≥ GluN2D; [290,365]). The receptor is also allosterically modulated, in both positive and negative directions, by endogenous neuroactive steroids in a subunit dependent manner [153,239]. Tonic proton blockade of NMDA receptor function is alleviated by polyamines and the inclusion of exon 5 within GluN1 subunit splice variants, whereas the non-competitive antagonists ifenprodil and traxoprodil increase the fraction of receptors blocked by protons at ambient concentration. Inclusion of exon 5 also abolishes potentiation by polyamines and inhibition by Zn 2+ that occurs through binding in the ATD [364]. Ifenprodil, traxoprodil, haloperidol, felbamate and Ro 8-4304 discriminate between recombinant NMDA receptors assembled from GluN1 and either GluN2A, or GluN2B, subunits by acting as selective, non-competitive, antagonists of heterooligomers incorporating GluN2B through a binding site at the ATD GluN1/GluN2B subunit interface [187]. LY233536 is a competitive antagonist that also displays selectivity for GluN2B over GluN2A subunitcontaining receptors. Similarly, CGP61594 is a photoaffinity label that interacts selectively with receptors incorporating GluN2B versus GluN2A, GluN2D and, to a lesser extent, GluN2C subunits. TCN 201 and TCN 213 have recently been shown to block GluN2A NMDA receptors selectively by a mechanism that involves allosteric inhibition of glycine binding to the GluN1 site [29,101,136,248]. In addition to influencing the pharmacological profile of the NMDA receptor, the identity of the GluN2 subunit co-assembled with GluN1 is an important determinant of biophysical properties that include sensitivity to block by Mg 2+ , single-channel conductance and maximal open probablity and channel deactivation time [74, 105,123]. Incorporation of the GluN3A subunit into tri-heteromers containing GluN1 and GluN2 subunits is associated with decreased single-channel conductance, reduced permeability to Ca 2+ and decreased susceptibility to block by Mg 2+ [52,142]. Reduced permeability to Ca 2+ has also been observed following the inclusion of GluN3B in tri-heteromers. The expression of GluN3A, or GluN3B, with GluN1 alone forms, in Xenopus laevis oocytes, a cation channel with unique properties that include activation by glycine (but not NMDA), lack of permeation by Ca 2+ and resistance to blockade by Mg 2+ and NMDA receptor antagonists [56]. The function of heteromers composed of GluN1 and GluN3A is enhanced by Zn 2+ , or glycine site antagonists, binding to the GluN1 subunit [236]. Zn 2+ also directly activates such complexes. The co-expression of GluN1, GluN3A and GluN3B appears to be required to form glycine-activated receptors in mammalian cell hosts [339]. AMPA and Kainate receptors All AMPA receptors are additionally activated by kainate (and domoic acid) with relatively low potency, (EC 50˜1 00 μM). Inclusion of TARPs within the receptor complex increases the potency and maximal effect of kainate [165,260]. AMPA is weak partial agonist at GluK1 and at heteromeric assemblies of GluK1/GluK2, GluK1/GluK5 and GluK2/GluK5 [171]. Quinoxalinediones such as CNQX and NBQX show limited selectivity between AMPA and kainate receptors. Tezampanel also has kainate (GluK1) receptor activity as has GYKI53655 (GluK3 and GluK2/GluK3) [171]. ATPO is a potent competitive antagonist of AMPA receptors, has a weaker antagonist action at kainate receptors comprising GluK1 subunits, but is devoid of activity at kainate receptors formed from GluK2 or GluK2/GluK5 subunits. The pharmacological activity of ATPO resides with the (S)-enantiomer. ACET and UBP310 may block GluK3, in addition to GluK1 [13,299].

IP 3 receptor
Ligand-gated ion channels → IP 3 receptor Overview: The inositol 1,4,5-trisphosphate receptors (IP 3 R) are ligand-gated Ca 2+ -release channels on intracellular Ca2+ store sites (such as the endoplasmic reticulum). They are responsible for the mobilization of intracellular Ca 2+ stores and play an important role in intracellular Ca 2+ signalling in a wide variety of cell types. Three different gene products (types I-III) have been isolated, which assemble as large tetrameric structures. IP 3 Rs are closely associated with certain proteins: calmodulin ( CALM1 CALM2 CALM3, P62158) and FKBP (and calcineurin via FKBP). They are phosphorylated by PKA, PKC, PKG and CaMKII.

Nicotinic acetylcholine receptors
Ligand-gated ion channels → Nicotinic acetylcholine receptors Overview: Nicotinic acetylcholine receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABA A , strychnine-sensitive glycine and 5-HT 3 receptors [6,257,334,350,387]. All nicotinic receptors are pentamers in which each of the five subunits contains four α-helical transmembrane domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ) have been identified [185]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [257]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). nAChRs contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the four transmembrane domains [124,401]; see also [144]). The high resolution crystal structure of the molluscan acetylcholine binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g. [53]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94 Å resolution [82], has revealed the orthosteric binding site in detail (reviewed in [55,185,315,334]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1) 2 β1δ , whereas an extrajunctional (α1) 2 β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and β(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [127,128,257]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4) 2 (β2) 2 or (α4) 3 (β2) 2 ] which influences the biophysical and pharmacological properties of the receptor [257]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [257] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [391]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10).
For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [257]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [9,183,256]). The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally-and/or heterologously-expressed nACh receptor subtypes [230]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses -see also [71] Functional Characteristics -(α9) 5 : P Ca /P Na = 9; α9α10: P Ca /P Na = 9, P f = 22% α9α10: P Ca /P Na = 9, P f = 22% -Comments Ligand interaction data for hetero-oligomeric receptors containing the β1 receptors are listed under the α1 receptors.
Comments: Commonly used agonists of nACh receptors that display limited discrimination in functional assays between receptor subtypes include A-85380, cytisine, DMPP, epibatidine, nicotine and the natural transmitter, acetylcholine (ACh). A summary of their profile across differing receptors is provided in [128] and quantitative data across numerous assay systems are summarized in [177]. Quantitative data presented in the table for commonly used antagonists and channel blockers for human receptors studied under voltage-clamp are from [46,58,291,292,295,386]. Type I PAMs increase peak agonist-evoked responses but have lit-tle, or no, effect on the rate of desensitization of α7 nicotinic ACh receptors whereas type II PAMs also cause a large reduction in desensitization (reviewed in [384]).  [253,345], KN62 (Negative) [122,329], ivermectin (Positive) [283] Comments ------Effects of the allosteric modulators at P2X7 receptors are species-dependent.
Comments: A317491 and RO3 also block the P2X2:P2X3 heteromultimer [113,173]. NF449, A317491 and RO3 are more than 10-fold selective for P2X1 and P2X3 receptors, respectively. Agonists listed show selectivity within recombinant P2X receptors of ca. one order of magnitude. A804598, A839977, A740003 and A438079 are at least 10-fold selective for P2X7 receptors and show similar affinity across human and rodent receptors [93,95,149]. Several P2X receptors (particularly P2X1 and P2X3) may be inhibited by desensitisation using stable agonists (e.g. αβ-meATP); suramin and PPADS are non-selective antagonists at rat and human P2X1-3,5 and hP2X4, but not rP2X4,6,7 [45], and can also inhibit ATPase activity [72]. Ip 5 I is inactive at rP2X2, an antagonist at rP2X3 (pIC 50 5.6) and enhances agonist responses at rP2X4 [199]. Antagonist potency of NF023 at recombinant P2X2, P2X3 and P2X5 is two orders of magnitude lower than that at P2X1 receptors [342]. The P2X7 receptor may be inhibited in a non-competitive manner by the protein kinase inhibitors KN62 and chelerythrine [329], while the p38 MAP ki-nase inhibitor GTPγS and the cyclic imide AZ11645373 show a species-dependent non-competitive action [94,253,254,345]. The pH-sensitive dye used in culture media, phenol red, is also reported to inhibit P2X1 and P2X3 containing channels [200].

ZAC
Ligand-gated ion channels → ZAC Overview: The zinc-activated channel (ZAC, nomenclature as agreed by the NC-IUPHAR Subcommittee for the Zinc Activated Channel) is a member of the Cys-loop family that includes the nicotinic ACh, 5-HT 3 , GABA A and strychnine-sensitive glycine receptors [78,155,363]. The channel is likely to exist as a homopentamer of 4TM subunits that form an intrinsic cation selective channel equipermeable to Na + , K + and Cs + , but impermeable to Ca 2+ and Mg 2+ [363]. ZAC displays constitutive activity that can be blocked by tubocurarine and high concentrations of Ca 2+ [363]. Although denoted ZAC, the channel is more potently activated by protons and copper, with greater and lesser efficacy than zinc, respectively [363]. ZAC is present in the human, chimpanzee, dog, cow and opossum genomes, but is functionally absent from mouse, or rat, genomes [78,155].

Comments:
The ZAC subunit does not appear to exist in the mouse or rat genomes [78]. Although tabulated as an antagonist, it is possible that tubocurarine acts as a channel blocker. Antagonism by Ca 2+ is voltage-independent. ZAC is not activated (at 1 mM) by transition metals including Fe 2+ , Co 2+ , Ni 2+ , Cd 2+ , or Al 3+ [363]. The concentration response relationship to Cu 2+ is biphasic, with concentrations exceeding 30 μM being associated with reduced activation [363].